Computational Structural Biology group focusing on dissecting, understanding and predicting biomolecular interactions at the molecular level.

Supported by:

The Docking

• Before starting
• Topologies and structures generation
• Rigid body energy minimization
• Semi-flexible simulated annealing
• Flexible refinement in explicit solvent (water or DMSO)

Before starting

Before starting the docking, you need to specify a number of parameters, such as:
• Number of structures to generate and refine
• Histidine protonation state
• Flexible segments
• Which kind of restraints to use and associated parameters
• Solvated docking options
• Scoring scheme
• Electrostatic treatment
• ...
Many of those have default values which you do not need to change.

Using your web browser, go to the HADDOCK online page, and use the Edit your run.cns option, choose the run.cns file to edit and click on "edit file". A new window will be created. (If the window is empty, try using a different browser).

For a description of the run.cns file, refer to the "run.cns file" section.

After setting all the parameters save the file as "run.cns" in your run directory using the "save updated file" button.

Note: If you have turned on the use of DNA/RNA restraints in run.cns HADDOCK expects to find a file called dna-rna_restraints.def in the data/sequence directory. This files allows you to define standard A-, B- or custom restraints for DNA such as base-pairing, puckering and backbone dihedral angles. You can edit a template file that can be found in the protocols directory and save it as dna-rna_restraint.def into the data/sequence directory. Using your web browser, go to the HADDOCK online page, and use the Edit your run.cns option, choose the dna-rna_restraints.def file to edit and click on "edit file". A new window will be created. (If the window is empty, try using a different browser).

When all necessary files and parameters have been properly edited and saved then start HADDOCK in the run directory by typing:
    haddock2.2

The entire protocol consists of four stages:

1. Topologies and structures generation

The first step in HADDOCK in the generation of the CNS topologies and coordinates files for the various molecules and for the complex from the input PDB files (see section PDB files). HADDOCK should automatically recognize chain breaks, disulphide bonds, cis-prolines and even ions provided they are named as defined in the ion.top topology file located in the toppar directory.

Job files will be generated in the run directory and the topologies, structures and output files will be generated in the begin directory. HADDOCK will use the fileroot names specified in the run.cns file.

The following scripts will be run:

• fileroot_generate_A/B/C/D/E/F.job: Generates the CNS topology and coordinates file(s) (if starting from an ensemble) for the various molecules.

Output files:
• fileroot_A/B/C/D/E/F.psf (topology)
• fileroot_A/B/C/D/E/F.pdb (coordinates)
• fileroot_A/B/C/D/E/F_1.pdb, fileroot_A/B/C/D/E/F_2.pdb, ... (if starting from an ensemble)
• file_A/B/C/D/E/F.list (list of PDB coordinates files)

CNS scripts called (depending on the options defined):
• generate_A/B/C/D/E/F.inp
• dna_break.cns
• dna-rna_restraints.def
• flex_segment_back.cns
• iterations.cns
• prot_break.cns
• run.cns
Note: If solvated docking is turned on, generate_A/B/C/D/E/F-water.inp will be used instead which calls in addition generate_water.cns,rotate_pdb.cns and generates additional output pdb files containing the water (fileroot_A/B/C/D/E/F_1_water.pdbw, ...)

• fileroot_generate_complex.job: Generates the CNS topology and coordinates file(s) for the complex by merging the various topologies and coordinates files. When starting from ensembles, all combinations will be generated.

Output files:
• fileroot.psf (topology)
• fileroot.pdb (coordinates)
• fileroot_1.pdb, fileroot_2.pdb, ... (if starting from an ensemble)
• file.cns, file.list, file.nam (list of PDB coordinates files)

CNS scripts called:
• generate_complex.inp
• iterations.cns
• run.cns
• separate.cns

Note: If solvated docking is turned on, generate_complex-water.inp will be used instead which will generates additional output pdb files containing the water (fileroot_1_water.pdbw ,...)
In case of problems (and in general to make sure that everything is OK) look into the output files generated (.out) for error messages (search for ERR).

2. Randomization and rigid body energy minimization:

The first docking step in HADDOCK is a rigid body energy minimization.

First the molecules will be separated by a minimum of 25A and rotated randomly around their center of mass. This randomization step can be turned off in the run.cns parameter file. If you wish to decrease (or increase) the separation distance between the two molecules, edit in the protocols directory the random_rotations.cns CNS script and change the value of the \$minispacing parameter.
The rigid body minimization is performed stepwise:
• four cycles of rotational minimization in which each molecule (molecule+associated solvent in case of solvated docking) is allowed to rotate in turn
• two cycles of rotational and translational rigid body minimization in which each molecule+associated solvent is treated as one rigid body

If solvated docking is turned on the following additional steps will be performed:
• rotational and translational rigid bogy minimization with each molecule and water molecule treated as separate rigid bodies

• Biased Monte Carlo removal of water molecules based on propensity of finding a water mediated contact until a user-defined percentage of water molecules remains

• rotational and translational rigid bogy minimization with each molecule and water molecule treated as separate rigid bodies

For details of the solvated docking protocol refer to:

If RDC or diffusion anisotropy restraints are used two additional minimization steps are carried out to optimize the orientation of the molecules with respect to the alignment tensor(s).
For each starting structure combination, this step can be repeated a number of times (given by the Ntrials parameter in the run.cns parameter file, and only the best solution is written to disk.

A new option in HADDOCK 2.x allows to systematically sample 180 degree rotated solutions. Since in our experience symmetrical solutions occur quite often, sampling of 180 degree rotated solutions can increase the success rate significantly. This option can be turned on and off with the rotate180_0 parameter in the run.cns parameter file.

Note: The translational minimization can be turned off in run.cns. This option can be useful for example for small flexible molecules to perform the docking during the simulated annealing stage allowing conformational changes to take place during the docking process. The number of steps in the first two stages of the simulated annealing should then be increased by at least a factor four to allow the molecules to approach each other.

The refine.inp CNS script is used for this step.
Output files:

• fileroot_1.pdb, fileroot_2.pdb, ... written in the structures/it0 directory

Note1: If solvated docking is turned on (waterdock=true in run.cns), additional output pdb files will be written to disk containing the water (fileroot_1_water.pdbw ,...).

Note2: If random removal of restraints is turned on (noecv=true in run.cns), additional files will be written to disk containing the random number seed (fileroot_1.seed ,...). This seed is used in the refinement to make sure that the same restraints are removed.

Note3: If random AIR definition is turned on (ranair=true in run.cns), additional files will be written to disk containing the list of residues selected for the AIR definition (fileroot_1.disp ,...)..

CNS scripts called in sequential order (depending on the options selected):
When all structures have been generated (typically in the order of 1000 to 2000 depending on the number of starting conformations and your CPU resources), HADDOCK will sort them accordingly to the criterion defined in the run.cns parameter file and write the sorted PDB filenames into file.cns, file.list and file.nam in the structures/it0 directory. These will be used for the next step (semi-flexible simulated annealing).

3. Semi-flexible simulated annealing

The best XXX structures after rigid body docking (typically 200, but this is left to the user's choice (see the run.cns file section)) will be subjected to a semi-flexible simulated annealing (SA) in torsion angle space. This semi-flexible annealing consists of several stages:
• High temperature rigid body search
• Rigid body SA
• Semi-flexible SA with flexible side-chains at the interface
• Semi-flexible SA with fully flexible interface (both backbone and side-chains)

The temperatures and number of steps for the various stages are defined in the run.cns parameter file.

Note1: HADDOCK also allows the definition of fully flexible regions (defined by the nfle_X parameter in run.cns) that remain fully flexible throughout all four stages. This should be useful for cases where part of a structure are disordered or unstructured or when docking small flexible ligands or peptides onto a protein. This option also allows the use of HADDOCK for structure calculations of complexes when classical NMR restraints are available to drive the folding.

Note2: A new option in HADDOCK 2.x allows to automatically define the semi-flexible regions by considering all residues within 5A of another molecule. To use this option, set nseg_X to -1 in run.cns (or another negative number if you still want to define manually segments for random AIRs definition from a limited region of the surface). This can be set for each molecule separately.

The refine.inp CNS script is used for this step.
Output files:

• fileroot_1.pdb, fileroot_2.pdb, ... written in the structures/it1 directory

Note1: If solvated docking is turned on (waterdock=true in run.cns), additional output pdb files will be written to disk containing the water (fileroot_1_water.pdbw ,...).

Note2: If random removal of restraints is turned on (noecv=true in run.cns), additional files will be written to disk containing the random seed number (fileroot_1.seed ,...). This seed is used in the explicit solvent refinement to make sure that the same restraints are removed.

CNS scripts called in sequential order (depending on options selected):
 initialize.cns iterations.cns run.cns read_struc.cns covalions.cns flags_new.cns read_data.cns calc_free-ene.cns read_water1.cns water_rest.cns flags_new.cns contactairs.cns water_rest.cns symmultimer.cns cm-restraints.cns surf-restraints.cns dna-rna_restraints.def set_noe_scale.cns mini_tensor.cns mini_tensor_dani.cns scale_inter_only.cns rotation180.cns flags_new.cns flex_segment_back.cns torsiontop.cns - flex_segment.cns - numtrees.cns sa_ltad_hightemp.cns - set_noe_scale.cns - scale_inter.cns sa_ltad_cool1.cns - set_noe_scale.cns - scale_inter.cns torsiontop_flex.cns - flex_segment_side.cns - numtrees.cns sa_ltad_cool2.cns - set_noe_scale.cns - scale_inter.cns torsiontop_flex_back.cns - flex_segment_back.cns - numtrees.cns sa_ltad_cool3.cns - set_noe_scale.cns - scale_inter.cns set_noe_scale.cns flex_segment_back.cns scale_intra_only.cns scale_inter_only.cns symmultimer.cns read_noes.cns dna-rna_restraints.def print_coorheader.cns waterdock_out1.cns
At the end of the calculation, HADDOCK generates the file.cns, file.list and file.nam files containing the filenames of the generated structures sorted accordingly to the criterion defined in the run.cns parameter file.
At the end of this stage, the structures are analyzed and the results are placed in the structures/it1/analysis directory (see the analysis section).

4. Flexible explicit solvent refinement

In this final step, the structures obtained after the semi-flexible simulated annealing are refined in an explicit solvent layer (8A for water, 12.5A for DMSO). In this step, no spectacular changes are expected, however, the scoring of the various structures is improved.

The re_h2o.inp or re_dmso.inp CNS script is used for this step.
Output files:

• fileroot_1w.pdb, fileroot_2w.pdb, ... written in the structures/it1/water directory

Note1: The numbering of the structures from it1 is kept.

Note2: If keepwater is set to true in run.cns, additional output pdb files will be written to disk containing the water (fileroot_1_h2o.pdb ,...).

CNS scripts called in sequential order (depending on the option defined):
 initialize.cns iterations.cns run.cns read_struc.cns flags_new.cns calc_free-solv-ene.cns calc_free-ene.cns read_water1.cns generate_water.cns (or generate_dmso.cns) water_rest.cns symmultimer.cns set_noe_scale.cns dna-rna_restraints.def mini_tensor.cns mini_tensor_dani.cns flex_segment_side.cns set_noe_scale.cns flex_segment_back.cns set_noe_scale.cns scale_intra_only.cns scale_inter_only.cns symmultimer.cns read_noes.cns dna-rna_restraints.def print_coorheader.cns
At the end of the explicit solvent refinement, HADDOCK generates the file.cns, file.list and file.nam files containing the filenames of the generated structures sorted accordingly to the criterion defined in the run.cns parameter file.

Finally, the structures are analyzed and the results are placed in the structures/it1/water/analysis directory (see the analysis section).